EP0135045B1 - Plasmid und Verfahren zur Verwendung desselben - Google Patents

Plasmid und Verfahren zur Verwendung desselben Download PDF

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Publication number
EP0135045B1
EP0135045B1 EP19840108599 EP84108599A EP0135045B1 EP 0135045 B1 EP0135045 B1 EP 0135045B1 EP 19840108599 EP19840108599 EP 19840108599 EP 84108599 A EP84108599 A EP 84108599A EP 0135045 B1 EP0135045 B1 EP 0135045B1
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EP
European Patent Office
Prior art keywords
plasmid
subtilis
natto
base sequence
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
EP19840108599
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English (en)
French (fr)
Other versions
EP0135045A2 (de
EP0135045A3 (en
Inventor
Kawabe Haruhide
Mukai Hiromichi
Arimura Hirofumi
Suyama Tadakazu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mitsubishi Tanabe Pharma Corp
Original Assignee
Green Cross Corp Japan
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Filing date
Publication date
Application filed by Green Cross Corp Japan filed Critical Green Cross Corp Japan
Publication of EP0135045A2 publication Critical patent/EP0135045A2/de
Publication of EP0135045A3 publication Critical patent/EP0135045A3/en
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Publication of EP0135045B1 publication Critical patent/EP0135045B1/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to a circular plasmid comprising at least an alpha-amylase promoter derived from Bacillus subtilis (natto).
  • Plasmids which are extrachromosomal genes, have been widely used in recently developed genetic engineering techniques as a vector for use in vitro recombination of genes.
  • the in vitro genetic recombination technique has a wide application, e.g., in achieving useful microorganisms.
  • Escherichia coil, B. subtilis, yeast, etc. are mainly used as hosts in genetic engineering.
  • yeast and B. subtilis may be used more advantageously than E. coli, since the former excrete their products outside the cells.
  • a further advantage of the use of yeast and B. subtilis is that they are not harmful to humans nor parasitic to various animals and plants, thus providing a genetic operation system having a high safety.
  • An object of the present invention is to provide a plasmid vector permitting an efficient manifestation of genes introduced therein in host cells.
  • Another object of the present invention is to provide a plasmid vector of a high copy number.
  • Still another object of the present invention is to provide a plasmid vector which permits efficient production of various proteins in B. subtilis and B. subtilis (natto).
  • Yet another object of the present invention is to provide a plasmid vector which can grow in both gram-positive and gram-negative bacteria.
  • the present invention is based on the above finding.
  • the alpha-amylase promoter gene may be obtained from B. subtilis (natto) GC 161, which has bacterial characteristics similar to those of B. subtilis.
  • This strain which is particularly preferred as a source of an alpha-amylase promoter gene has been deposited at Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, on June 6, 1984 under deposition No. FERM-BP No. 539.
  • the alpha-amylase promoter gene can be isolated from the chromosomal DNA of GC 161 strain in a conventional method as described in, for example, Palva, I.: Gene 15. 43-51 (1981
  • the DNA thus obtained after having been digested into fragments with various known restriction enzymes, can be ligated with a known plasmid as an intermediate, and a-amylase gene-containing plasmids can be screened using starch-iodine reaction.
  • the base sequence of a-amylase gene DNA can be determined according to the method described by Maxam, A. and Gilbert, W.: Methods in Enzymology 65,499-560 to obtain the base sequence illustrated in Fig. 1.
  • region -214 to -1 can be used as a promoter. If desired, smaller region beginning with -200 can be used (Fig. 2).
  • promoter fragments can include a signal sequence i.e., base sequence of at most region 1 to 123 in addition to region -214 (or -200) to -1 (Fig. 3), if desired.
  • the a-amylase promoter containing plasmid of the present invention can be ligated with a plasmid vector derived from gram-negative bacteria such as E. coli (e.g., pBR 322) and further introduced in a plasmid vector derived from gram-positive bacterial as B. subtilis, B. subtilis (natto), Staphylococcus aureus, etc. (e.g., pUB 110) to obtain a shuttle vector containing an a-amylase promoter derived from B. subtilis (natto) and capable of growing in both gram-positive bacteria such as B. subtilis, B. subtilis (natto), etc. and gram-neqa t ive bacteria such as E. coli.
  • a plasmid vector derived from gram-negative bacteria such as E. coli (e.g., pBR 322)
  • a plasmid vector derived from gram-positive bacterial as B. subtilis, B. subtil
  • Various genes coding for useful physiologically active substances can be inserted into a position downstream of the a-amylase promoter and signal sequence, and the resulting plasmid vectors can be used in transforming B. subtilis, B. subtilis (natto), E. coli, etc. preferably B. subtilis, or B. subtilis (natto), and causing the transformed bacteria to express newly introduced genetic information, thus efficiently producing desired proteins such as various interferons, urokinase, HB vaccine, etc.
  • Digestion of the extrachromosonal and chromosonal DNA or ligation of fragments of such DNA can be performed by mixing a DNA solution having a final DNA concentration of 0.5 to 10 microgram/microliter, preferably 1 microgram/microliter, a suitable buffer solution and a restriction enzyme or a ligase in an amount of 1 to 10, preferably 3, units/microgram DNA, and allowing the mixture to stand generally at 10 to 40°C, preferably 37°C for 2 hours to overnight, preferably 3 to 4 hours.
  • Bcl I, Sma land Tag T ranges of reaction temperature used are 20 to 55°C (preferably 50°C), 5 to 35°C (preferably 30°C) and 30 to 70°C (preferably 65°C), respectively.
  • Fig. 1 The base sequence of the promoter and structural gene of a-amylase DNA is shown in Fig. 1.
  • a-Amylase gene was isolated from the chromosomal DNA of B. subtilis (natto) GC161 described above.
  • the isolation of the chromosomal DNA was performed according to a conventional method (Palva, I.: Gene 15, 43 ⁇ 51 (1981)) as follows.
  • Chromosomal DNA fraction was collected, which was removed of ethidium bromide with isoamyl alcohol in a conventional manner and dialyzed against TES solution followed by treatment with phenol twice to obtain purified chromosomal DNA.
  • the chromosomal DNA thus obtained was digested with restriction enzymes Pvu II, Bgl II, Hpa II, Bam HI, Hind III and subjected to electrophoresis on 0.8% agarose gel.
  • DNA fragments were extracted from the agarose gel and ligated with various known plasmids (pC 194, pE 194, pUB 110, and pGC 200 derived from pC 194) and transformed to B. subtilis strain (rec - , amy-).
  • the transformation was performed to spread on the L-agar plates containing both 1% soluble starch and an antibiotic, and incubated at 37°C for 2 days.
  • Iodine solution (0.1 % iodine - 1 % KI) was dropped onto the agar plate and decoloration of iodine which indicates presence of a-amylase activity was checked.
  • a strain having plasmid pGC 301 (15 kb) which was obtained by inserting a DNA fragment of B. subtilis (natto) GC 161 digested with Hind III to pGC 200 (8 kb) at its Hind III position, showed a-amylase activity.
  • the pGC 301 was digested with Hind III completely and with Rsa I partially, and loaded on 0.8% agarose gel.
  • the DNA fragments of various sizes obtained from agarose gel with ligated with a Eco RV-Hind fragment of pBR 322.
  • pGC 302 (10 Kb) was obtained.
  • pGC 302 was digested with Suc II and a 7.9 Kb DNA fragment was isolated and ligated.
  • pGC 303 was prepared (Fig. 4).
  • pGC 303 (7.9 kb) containing promotor, signal sequence and structural gene for a-amylase derived from B. subtilis (natto) pGC 161 was prepared (Fig. 1).
  • pGC 303 is a plasmid whose host is E. coli.
  • the pGC 303 obtained in Example 1 was ligated with pGC 18 as obtained in the following Example 3 and deposited at Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, on July 6, 1983 under deposition No. FERM-BP No. 312 (named B. subtilis (natto) GC 18), or pUB 110, derived from Staphylococcus aureus in a conventional manner.
  • pGC 303 was digested with Bam HI and Pvu II and pUB 110 was also digested with the same restriction enzymes. DNA fragments obtained were ligated with each other using T 4 ligase to prepare a hybrid plasmid pGC 304 having a molecular weight of 10.7 kb.
  • pGC 303 was digested with Bam HI, Pvu III and Pol I while pGC 18 was digested with Pvu II, and DNA fragments obtained were ligated with each otherto give a hybrid plasmid pGC 305 having a molecular weight of 13.7 kb.
  • B. subtilis (natto) GC 18 strain was cultured in 500 ml of L broth (10 g of peptone, 5 g of yeast extract, 1 g of glucose and 5 g of NaCl, water to make 1 liter) at 30°C with shaking, and cells were harvested by centrifugation. The cells thus collected were suspended in 18 ml of 25% sucrose TES solution (TES solution: 30 mM Tris HCI (pH 8.0), 50 mM, NaCl, and 5 mM EDTA).
  • Polyethylene glycol was added to the supernatant to a final concentration of 10% and the mixture was allowed to stand overnight at 4°C, and subjected to centrifugation at 10,000 rpm for 10 minutes.
  • the residue was dissolved in TES and subjected to cesium chloride-ethidium bromide density gradient centrifugation at 60,000 rpm for 16 hours using a Beckmann Type 70.1 Ti rotor to obtain a plasmid DNA.
  • the plasmid DNA was washed with isoamy alcohol in a conventional mannerto remove ethidium bromide and dialyzed against 1,000 volumes of TES at 4°C.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Saccharide Compounds (AREA)
  • Enzymes And Modification Thereof (AREA)

Claims (6)

1. Ringförmiges Plasmid, welches mindestens einen von Bacillus subtilis (natto) stammenden alpha-Amylase-Promotor umfasst, wobei der alpha-Amylase-Promotor die in Fig. 2 aufgeführte Basensequenz aufweist.
2. Ringförmiges Plasmid nach Anspruch 1, wobei das Plasmid einen alpha-Amylase-Promotor und eine Signalsequenz, die von Bacillus subtilis (natto) stammen, umfasst und die in Fig. 3 dargestellte Basensequenz aufweist.
3. Ringförmiges Plasmid nach Anspruch 1, wobei das Plasmid im weiteren eine Plasmid-DNA-Basensequenz, die von E. coli stammt, umfasst.
4. Ringförmiges Plasmid nach Anspruch 3, wobei die Plasmid-DNA-Basensequenz eine partielle Basensequenz von pBR 322 darstellt.
5. Ringförmiges Plasmid nach Anspruch 3, wobei das Plasmid im weiteren eine Plasmid-DNA-Basensequenz, die von Bacillus subtilis (natto) oder Staphylococcus aureus stammt, umfasst.
6. Verwendung eines Plasmids nach den Ansprüchen 1 bis 5 zur Herstellung von Peptiden und Proteinen.
EP19840108599 1983-07-22 1984-07-20 Plasmid und Verfahren zur Verwendung desselben Expired EP0135045B1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP135032/83 1983-07-22
JP58135032A JPS6027388A (ja) 1983-07-22 1983-07-22 プラスミツド

Publications (3)

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EP0135045A2 EP0135045A2 (de) 1985-03-27
EP0135045A3 EP0135045A3 (en) 1987-01-28
EP0135045B1 true EP0135045B1 (de) 1990-12-19

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EP19840108599 Expired EP0135045B1 (de) 1983-07-22 1984-07-20 Plasmid und Verfahren zur Verwendung desselben

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EP (1) EP0135045B1 (de)
JP (1) JPS6027388A (de)
DE (1) DE3483760D1 (de)
ES (1) ES534842A0 (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020124830A1 (zh) * 2018-12-17 2020-06-25 江南大学 基于人工串联启动子的枯草芽孢杆菌高效诱导表达系统

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61135599A (ja) * 1984-12-04 1986-06-23 Green Cross Corp:The 異種蛋白質の製造法
JPS61177996A (ja) * 1985-02-02 1986-08-09 Green Cross Corp:The インタ−フエロン−γの製造法
JPH0779698B2 (ja) * 1985-10-01 1995-08-30 萬有製薬株式会社 安全性の高い多機能クロ−ニングベクタ−
JPH07108224B2 (ja) * 1985-11-07 1995-11-22 重三 鵜高 バチルス・ブレビスを用いる遺伝子の発現方法
GB8704908D0 (en) * 1987-03-03 1987-04-08 Apcel Ltd Vectors
JPH0335105U (de) * 1989-08-15 1991-04-05
JPH0486835U (de) * 1990-12-06 1992-07-28

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020124830A1 (zh) * 2018-12-17 2020-06-25 江南大学 基于人工串联启动子的枯草芽孢杆菌高效诱导表达系统
US12098373B2 (en) 2018-12-17 2024-09-24 Jiangnan University Bacillus subtilis efficiently-induced expression system based on artificial series promoter

Also Published As

Publication number Publication date
ES8602122A1 (es) 1985-12-01
DE3483760D1 (de) 1991-01-31
EP0135045A2 (de) 1985-03-27
EP0135045A3 (en) 1987-01-28
ES534842A0 (es) 1985-12-01
JPS6027388A (ja) 1985-02-12

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